Virological safety is a significant concern in the biopharmaceutical industry. Despite efforts to mitigate the risk, incidents involving large-scale viral contamination of biologics have raised concern in the industry. Highly profiled events include, for example, Genzyme's 2009 detection of a Vesivirus 2117 contamination of its CHO (Chinese hamster ovary) cell culture which halted production of Cerezyme® and Fabrazyme® and Merck's 2010 contamination of its Rotarix® vaccine by porcine circovirus 1. A likely source of contamination is at the cell culture stage. In addition to the economic toll on the manufacturing company (one report puts the estimate at over hundred million loss per 10000 L bioreactor contamination plus fines from the agencies), such events pose a risk to patients and disrupt access to the biopharmaceutical products (Liu et al., Biotechnol. Prog. 2000, 16, 425-434). As a result, there is heightened regulatory scrutiny and demand for new techniques to detect, prevent, and remediate viral contaminations.
In general, viral contaminants can be differentiated into upstream and downstream viral contaminations. Downstream contaminations may be controlled by applying closed systems, however, especially upstream contaminations are difficult to control and detect even by extensive testing. Viral contaminants may also originate from the use of animal derived materials in the biopharmaceutical production. Where the production cell line is free of extraneous viral contaminants and production does not involve use of animal derived materials, viral contaminants could still enter by way of cell culture media. For instance, synthetic media may be supplemented with recombinant growth factors produced in a serum-supplemented system and protein-free medium may nevertheless contain filtered protein hydrolysates. However, viral contamination may even occur in completely chemically defined medium, because large quantities of medium components may be packed in non-sterile containers. Conventional sterilizing-grade filters are neither designed to nor capable of safeguarding against viral contaminants, so other measures must be employed to ensure virological safety.
Detection of adventitious viruses at one or more checkpoints of the production process is standard practice. However, detection alone is an inadequate measure against viral contamination of biopharmaceutical products, especially where the viral contaminant present is unsuspected, unknown, or an emerging viral agent. Such viral agents can escape detection by even well-designed DNA microarrays representative of a large collection of sequenced viruses. The challenge is further compounded by the low levels of viral contaminants needed to infect a cell culture and currently limited detection assay sensitivity.
High titers of the viral contaminant may not manifest in the form of altered cell culture parameters, e.g. culture density, protein titers, beyond their normal range. On the other hand, infectivity assays are highly specific and require different conditions for each virus. As a result of viral contamination, downstream equipment, fluids, and products can be tainted, incurring millions of dollars in batch setup, waste disposal, lost sales, and decontamination. Thorough screening of raw materials for viruses is difficult due to sample heterogeneity and the large volumes involved in biopharmaceutical production processes.
Viral clearance techniques can be classified into one of two groups: inactivation and filtration. Inactivation methods seek the irreversible loss of viral infectivity, whereas filtration methods seek to mechanically reduce the viral contaminant. Conventional inactivation methods employ ultraviolet (UV) irradiation, gamma irradiation, heat, low or high pH, or solvent/detergent exposure. In instances where UV irradiation can effectively and irreversibly eliminate viral activity, it may be impractical on a large-scale basis or unsuitable for prepared media. Autoclaving, while possible for heat-stable liquids, may alter sensitive media. An alternative method known as high-temperature, short-time (HTST) heat treatment is not as harsh but demands costly equipment, automation, and validation procedures to preserve the media characteristics. Low or high pH exposure is ineffective across the spectrum of possible viral contaminants and can negatively impact the quality or osmolarity of the media. Solvent/detergent exposure is likewise not a broad-spectrum solution and is effective only for viruses with a lipid envelope. As such, the ideal method should balance cost considerations and the needs to effect viral clearance in raw materials and provide a broad-spectrum solution without compromising growth rate or yield.
Viral-retentive filtration offers the appropriate balance. It does not chemically alter media components and is suitable for use with heat-sensitive feed/media. Furthermore, viral-retentive filtration is a broad-spectrum solution since it operates on a size exclusion principle. However, viral-retentive membranes are costly (approximately about 2000 to 5000 EUR per m2). The low specific flow rates characteristic of filtration of media volumes have made the method economically taxing on a scale suitable for large scale bioreactor supply, due to the cost of the membrane area needed. For example, where virus filtration is connected in-series to sterilizing grade media filtration, virus filtration preferably needs to occur within a working day, i.e. a maximum of 2 to 10 hours after preparation of the bulk medium in order to prevent contamination of the bulk medium. Therefore, a large filtration area is needed to stay within this critical time window, which in turn raises costs.
Surprisingly, it has been found that the drawbacks of said prior art virus filtration can be overcome by filtration of the respective preparation, being a cell culture medium or at least a component of a cell culture medium, for at least about 24 hours through a virus filter having an effective pore size of maximum 75 nm. If the required volume of the respective preparations is filtered during a longer time frame, i.e. for at least 24 hours the volumetric capacity of the virus filters increase enormously. Surprisingly, it has been found additionally that significant overall virus titer reduction can be achieved over this extended period of time. This is especially beneficial in upstream virus removal in cell culture systems.
Therefore, the method of the invention enhances the economic efficiency of virus filtration by enhancing throughput and volumetric capacity, respectively. The method according to the present invention operates at a volumetric capacity of at least 2000 L/m2, thereby helping to maximize the use of high capacity virus filters, decreasing the effective costs associated therewith, and presenting a solution practicable on a large scale and readily integrable into existing production processes.
The enormous impact of the method according to the invention and the inventive use of the respective virus filters on sterile manufacturing processes, in particular processes where sterile preparations, e.g. cell culture media and buffers, are used can be understood by means of the following example. Assuming that a square meter of a virus filter membrane costs about 3000 EUR in average and a cell culture medium is used costing about 10 EUR per liter medium, then the costs for 1000 L virus filtered media are 13 EUR per liter medium, which increases the costs of goods for media preparation by about 30%. If 2000 L can be filtered with a virus filter membrane then the costs decrease to 11.50 EUR. Further increase of volumetric capacities, e.g. beyond 5000 L reduces the costs to less than 0.6 EUR per liter medium, which makes the additional costs for providing a virus filtered medium considerably low. As a result, the high costs of using virus filters, in particular in upstream decontamination of potential viral or viral contamination decreases significantly by increasing the volumetric capacity of the virus filtration method.
The present invention fully addresses this problem of high costs and low volumetric capacity of virus filters, respectively. The volumetric capacity of the virus filter can be increased by performing the virus filtration for at least about 24 hours through a virus filter having an effective pore size of maximum 75 nm. Surprisingly, it has been found that the volumetric capacity of the used costly virus filters can be better exploited leading to a 2 to 100-fold increase of the volumetric capacity while maintaining the filter integrity. Although a 2 to 3-fold increase of volumetric capacity already has a great impact to the production process and the related production costs, with the method according to the invention an up to 100-fold increase of volumetric capacity or even more can be achieved. This offers great opportunities and makes viral removal cost efficient even with costly virus filters that now can be used to further improve viral safety in cell culture processes, in particular in upstream viral removal of cell culture processes, pharmaceutical, diagnostic and/or cosmetic and food processes.